The invention relates to trophoblast cell preparations and uses of the cell preparations.
In mammals, the earliest developmental decision specifies the trophoblast cell lineage. In mice, this lineage appears at the blastocyst stage as the trophectoderm, a sphere of epithelial cells surrounding the inner cell mass (ICM) and the blastocoel. After implantation, the ICM gives rise to the embryo proper and some extraembryonic membranes. However, the trophectoderm is exclusively restricted to form the fetal portion of the placenta and the trophoblast giant cells. The polar trophectoderm (the subset of trophectoderm in direct contact with the ICM) maintains a proliferative capacity and gives rise to the extraembryonic ectoderm (ExE), the ectoplacental cone (EPC), and secondary giant cells of the early conceptus (1). The rest of the trophectoderm ceases to proliferate and becomes primary giant cells. Studies in primary culture and chimeric mice have suggested that stem cells exist in the extraembryonic ectoderm which contribute descendants to the EPC and the polyploid giant cells (2). Further evidence indicated that maintenance of these stem cell-like characteristics was dependent on signals from the ICM and later from the epiblast (3), since diploid trophoblast cells transformed into giant cells when removed from the embryonic environment (4). However, the nature of the embryo-derived signal was not known and all attempts at routine long-term culture of mouse trophoblast stem cells have been unsuccessful.
Expression and functional analyses indicated that Fgf4 and Fgfr2 may be involved in trophoblast proliferation (5, 6, 7). The reciprocal expression domains of Fgfr2 and Fgf4 suggested that the trophoblast could be a target tissue for an embryonic FGF signal. Fgfr2-null and Fgf4-null mice show similar peri-implantation lethal phenotypes (6, 7). This may result from defects in the ICM and its endoderm derivatives. However, it is also consistent with the possibility that FGF4 acts on the trophoblast through FGFR2 to maintain a proliferating population of trophoblast cells. Support for this latter possibility is provided by recent studies showing that inhibiting FGF signaling blocked cell division in both the ICM and trophectoderm (8).
The present inventors have found that FGF4 can promote sustained proliferation of primary cultures of diploid trophoblast cells and it permits isolation of stable FGF4-dependent mouse trophoblast stem (TS) cell lines from both the ExE of 6.5 dpc embryos and the trophectoderm of 3.5 dpc blastocysts. TS cell lines expressed many diploid trophoblast markers and retained the capacity to differentiate into other trophoblast subtypes in vitro upon removal of FGF4. Most importantly, when these stem cells were introduced into chimeras they exclusively contributed to all trophoblast subtypes in vivo. Availability of trophoblast stem cell lines opens up new possibilities for understanding the genetic regulation of placental development and placental insufficiencies and modulating the same. The cell lines also enable the treatment of placental insufficiencies by pharmacological intervention or gene-based therapy.
Broadly stated, the present invention relates to a stable pluripotent trophoblast stem (TS) cell line. In particular, the invention relates to a purified preparation of trophoblast stem cells which (i) arc capable of indefinite proliferation in vitro in an undifferentiated state; and (ii) are capable of differentiation into cells of the trophoblast lineage in vivo. The preparation of trophoblast stem cells is also characterized by expression of genetic markers of diploid trophoblast stem cells.
A trophoblast stem cell preparation of the invention may be induced to differentiate into cells of the trophoblast lineage in vitro or in vivo. The invention therefore also relates to a purified trophoblast stem cell preparation of the invention (preferably cultured in vitro) induced to differentiate into cells of the trophoblast lineage. This differentiated cell preparation is characterized by expression of genetic markers of trophoblast cell lineages (e.g. diploid trophoblast cells of the ectoplacental cone (EPC), and the secondary giant cells of the early conceptus). In an embodiment of the invention a purified trophoblast cell preparation comprises cells of the trophoblast lineage including diploid trophoblast cells.
A cell preparation of the invention may be derived from or comprised of cells that have been genetically modified either in nature or by genetic engineering techniques in vivo or in vitro.
Cell preparations or cell lines of the invention can be modified by introducing mutations into genes in the cells or by introducing transgenes into the cells. Insertion or deletion mutations may be introduced in a cell using standard techniques. A transgene may be introduced into cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection. Suitable methods for transforming and transfecting cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks. By way of example, a transgene may be introduced into cells using an appropriate expression vector including but not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses). Transfection is easily and efficiently obtained using standard methods including culturing the cells on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. 6:383-388).
A gene encoding a selectable marker may be integrated into cells of a cell preparation of the invention. For example, a gene which encodes a protein such as xcex2-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or a fluorescent protein marker may be integrated into the cells. Examples of fluorescent protein markers are the Green Fluorescent Protein (GFP) from the jellyfish A. victoria, or a variant thereof that retains its fluorescent properties when expressed in vertebrate cells. (Examples of GFP variants include a variant of GFP having a Ser65Thr mutation of GFP (S65T) that has longer wavelengths of excitation and emission, 490 nm and 510 nm, respectively, compared to wild-type GFP (400 nm and 475 nm): a blue fluorescent variant of GFP (e.g. N66H-GFP) (Heim et al. Proc. Natl. Acad. Sci. 91:12501, 1994). MmGFP (M. Zernicka-Goetz et al, Development 124:1133-1137, 1997), enhanced GFP (xe2x80x9cEGFPxe2x80x9d) (Okabe, M. et al. FEBS Letters 407:313-319, 1997; Clontech Palo Alto, Calif.), EGFP which has a Phe to Leu mutation at position 64 resulting in the increased stability of the protein at 37xc2x0 C. and a Ser to Thr mutation at position 65 resulting in an increased fluorescence, and, EGFP commercially available from Clontech incorporating a humanised codon usage rendering it xe2x80x9cless foreignxe2x80x9d to mammalian transcriptional machinery and ensuring maximal gene expression.)
The invention also relates to a method for producing a purified trophoblast stem (TS) cell preparation i.e. a cell line, comprising the steps of culturing early postimplantation trophoblast cells or cells of a blastocyst, preferably from the trophectoderm on a feeder layer (e.g. a fibroblast layer or a medium conditioned by fibroblasts) in the presence of FGF4 and a co-factor. The method may additionally comprise inducing differentiation of the trophoblast stem cells by removing the FGF4, the co-factor, or the feeder layer. In an embodiment of the invention, the method comprises isolating a blastocyst, culturing the blastocyst on a fibroblast layer in the presence of FGF4 and a co-factor, removing a blastocyst outgrowth and dissociating the outgrowth, selecting flat colonies i,e. epithelial-like cells, and culturing the colonies. The invention also contemplates trophoblast cell preparations or lines derived at all stages of development under the same culture conditions.
The term xe2x80x9cblastocystxe2x80x9d used herein refers to the structure during early embryonic development comprising an inner cluster of cells, the inner cell mass (ICM), which gives rise to the embryo, and an outer layer, the trophectoderm, which gives rise to extra-embryonic tissues. Preferably, cells from the trophectoderm of a 3.5 dpc blasotocyst are used in the method of the invention. The term xe2x80x9cpostimplantation trophoblastsxe2x80x9d used herein refers to cells derived from extraembryonic extoderm (ExE) cells preferably isolated from 6.5 days post coitum conceptuses. The term xe2x80x9cepithelial-like cellsxe2x80x9d refers to the flat colonies obtained after dissociation of a blastocyst outgrowth and which are like the cells which sometimes appear during the isolation of embryonic stem cells from blastocysts as described in B. Hogan et al (10).
The blastocysts or early postimplantation trophoblasts may be derived or isolated from any mammalian or marsupial species including but not limited to rodents (e.g. mouse, rat, hamster, etc.), rabbits, sheep, goats, pigs, cattle, primates, and humans are preferred. Mutant or transgenic blastocysts and postimplantation trophoblasts may be used to prepare a cell preparation or cell line of the invention. For example, a cell preparation or cell line of the invention may be derived from a Fgf4 or Errxcex2 mutant blastocyst. Cells used to prepare a cell preparation or cell line of the invention can be engineered to contain a selectable marker or they may be genetically altered using techniques well known in the art.
The cells derived from a blastocyst or postimplantation trophoblast cells are cultured on a feeder layer. The feeder layer may be a confluent fibroblast layer, preferably primary mouse embryonic fibroblast (EMFI) cells. Embryonic fibroblasts may be obtained from 12 day old fetuses from outbred mice, but other strains may be used as an alternative. STO cells (i.e. a permanent line of irradiated mouse fibroblasts) can also be used as a feeder layer. The feeder layer may also comprise medium conditioned by primary embryonic fibroblast cells.
Cells from a blastocyst or early postimplantation trophoblast cells are preferably cultured in medium comprising RPMI 1640 with 20% fetal bovine serum, sodium pyruvate, xcex2-mercaptoethanol, L-glutamine, and penicillin/streptomycin. The FGF4 used in the method of the invention may be recombinant FGF4 (preferably recombinant human FGF4) which may be produced using standard recombinant techniques or it may be obtained from commercial sources (e.g. Sigma). The co-factor used in the method of the invention is preferably heparin. Once established the cell lines may be grown on a feeder layer such as a fibroblast layer (e.g. EMFI cells) or in a conditioned medium prepared from a fibroblast layer (See for example the medium described in note 13, page 15).
Cells from the cell preparations may be introduced into a blastocyst or aggregated with an early stage embryo to produce chimeric conceptuses. A chimeric conceptus may be allowed to grow to term, or sacrificed during gestation to observe the contribution of the stem cell line. In an embodiment, the invention provides a chimeric placenta wherein the trophoblast lineage is repopulated by cells from a cell preparation of the invention. The conceptuses and placenta can be engineered to carry selectable markers or genetic alterations. Cell lines can be derived from the chimeric conceptuses and placenta. Therefore, the invention further provides a chimeric conceptus, differentiated trophoblast cells, mutant trophoblast stem cells, or a chimeric placenta derived from a purified preparation of the invention.
The cell preparations, chimeric conceptuses, and chimeric placentas may be used to screen for potential therapeutics that modulate trophoblast development or activity e.g. Invasion or proliferation. In particular, the cell preparations, chimeric embryos, or chimeric placenta may be subjected to a test substance, and the effect of the test substance may be compared to a control (e.g. in the absence of the substance) to determine if the test substance modulates trophoblast development or activity. Cell preparations of the invention derived from mouse mutants can be used to identify genes and substances that are important for the trophoblast cell lineage, and in vitro differentiation of mutant cell preparations can identify genes and substances important for selected trophoblast subtypes. Selected substances may be useful in regulating trophoblasts in vivo and they may be used to treat various conditions requiring regulation of trophoblast development or activity such as the conditions described below.
The cell preparations of the invention may be transplanted into animals to treat specific conditions requiring modulation of trophoblast development or activity. For example, the cell preparations may be used to prolong fetal survival in conditions of placental insufficiency, or to reduce uncontrolled trophoblast invasion and abnormal trophoblast growth associated with conditions such as hydatiform mole and choriocarcinoma. The cell preparations may be used for therapeutic treatment of placental defects in humans by transplantation of the cell preparations at any stage of pregnancy to generate chimeric placenta.
The cell preparations may be used to prepare model systems of disease for conditions such as precclampsia, hydatiform mole, or choriocarcinoma.
The cell preparations or cell lines of the invention can be used to produce growth factors, hormones, etc. relevant to human placenta. The cell preparations or cell lines of the invention can also be used to produce therapeutics such as human Chorionic Gonadotropin (hCG).
The cell preparations or cell lines of the invention can be used to screen for genes expressed in or essential for trophoblast differentiation. Screening methods that can be used include Representational Difference Analysis (RDA) or gene trapping with for example SA-lacZ (D. P. Hill and W. Wurst, Methods in Enzymology, 225: 664, 1993). Gene trapping can be used to induce dominant mutations (e.g. by deleting particular domains of the gene product) that affect differentiation or activity of trophoblast cells and allow the identification of genes expressed in or essential for trophoblast differentiation.